WO2015109952A1 - 一种人源抗虫基因及其编码的抗Cry1B毒素独特型单链抗体与应用 - Google Patents

一种人源抗虫基因及其编码的抗Cry1B毒素独特型单链抗体与应用 Download PDF

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WO2015109952A1
WO2015109952A1 PCT/CN2015/070421 CN2015070421W WO2015109952A1 WO 2015109952 A1 WO2015109952 A1 WO 2015109952A1 CN 2015070421 W CN2015070421 W CN 2015070421W WO 2015109952 A1 WO2015109952 A1 WO 2015109952A1
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Prior art keywords
human
chain antibody
cry1b
resistant gene
toxin
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PCT/CN2015/070421
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English (en)
French (fr)
Chinese (zh)
Inventor
刘贤金
徐重新
张霄
刘媛
谢雅晶
张存政
余向阳
王冬兰
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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Priority to US15/104,127 priority Critical patent/US9770038B2/en
Priority to JP2016535243A priority patent/JP6196382B2/ja
Priority to BR112016016014-2A priority patent/BR112016016014B1/pt
Priority to EP15739989.0A priority patent/EP3098316B1/en
Publication of WO2015109952A1 publication Critical patent/WO2015109952A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4233—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Gram-positive bacteria
    • C07K16/1278—Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • the invention relates to the field of genetic engineering and biological control, in particular to a human-resistant insect-resistant gene and an encoded anti-Cry1B toxin unique type single-chain antibody and application thereof.
  • Bacillus thuringiensis Bacillus thuringiensis
  • Bacillus thuringiensis Bacillus thuringiensis is an insect pathogenic bacterium, and its Bt toxin has a specific poisoning effect on a variety of agricultural and forest pests.
  • Plant Genetic Systems of Belgium reported the success of transgenic Bt insect-resistant tobacco for the first time in the world.
  • Bt gene has been transferred to corn, rice, cotton, tomato, potato, tobacco, etc. Among the world's major crops.
  • Bt toxin protein In the liver and kidney, cell swelling and vacuolar-like degeneration can be observed, and Seeing the lesions of glomerular vascular epithelial cells, of course, this cannot be ruled out by the immune response. Long-term high-dose use of Bt toxin protein also resulted in a significant decrease in the total number of white blood cells and hemoglobin in animals, indicating that Bt toxin protein has significant immunosuppressive toxicity. The development of alternative biological effectors with Bt toxin bioactivity (eg anti-idiotypic antibodies) is a hot spot in the development of biopesticide research.
  • the humanized antibody gene has the advantage of being homologous to the human immune system gene because it is derived from human, and can avoid the damage to the human immune system caused by the residue in the food after its preparation spray or transgenic expression.
  • An anti-idiotype antibody (hereinafter referred to as Anti-Id) is a specific antibody produced by a unique type (Idiotype, Id) located in the variable region of an antibody molecule.
  • Id an anti-idiotype antibody
  • Bona et al. classify anti-idiotypic antibodies into four types ( ⁇ , ⁇ , ⁇ , and ⁇ ) based on the serological response of Id and Anti-Id and the function of AId.
  • the ⁇ -type anti-idiotypic antibody has an antigenic function and biological activity because it has an "inner image" effect, that is, has the same antigenic determinant as the (semi)antigen.
  • phage display technology can be used to construct an anti-idiotypic antibody with a similar effect of a target antigen by constructing a phage antibody library.
  • the process of screening for specific antibodies using phage display technology is called “Panning” and consists of four steps of binding, washing, elution and amplification.
  • Raats et al. used anti-cortisol monoclonal anti-coating as a solid phase antigen for direct screening, and negative screening with negative monoclonal antibodies of the same species before screening to avoid screening for recombinant antibody fragments that bind to the constant region of the antibody.
  • the anti-Id against cortisol was successfully screened. Goletz et al.
  • a human insect-resistant gene having a nucleotide sequence as shown in SEQ ID NO.
  • the anti-Cry1B toxin idiotype single-chain antibody encoded by SEQ ID NO. 1 has an amino acid sequence as shown in SEQ ID NO.
  • a prokaryotic expression vector comprising the human insect resistance gene SEQ ID NO.
  • a human insect resistance gene SEQ ID NO. 1 is used in the control of agricultural pests.
  • an insecticide comprising an anti-Cry1B toxin idiotype single-chain antibody having an amino acid sequence as shown in SEQ ID NO.
  • the present invention screens and obtains a " ⁇ " type anti-Cry1B toxin unique type single-chain antibody having insecticidal activity from the disclosed human gene library, and the single chain antibody is expressed by a prokaryotic system, and the primary culture has a vertical volume with rice.
  • the binding activity of the peritrophic membrane specific receptor BBMV in the midgut of the leaf mites the invention obtains the " ⁇ " type anti-Cry1B toxin unique type single-chain antibody without animal immunization, has a short preparation cycle, small amino acid sequence, and is suitable for large-scale in vitro.
  • the present invention as a brand new new insect-resistant genetic resource, explores and expands new insect-resistant genetic resources with simulated Bt toxin biological activity, reduces various safety risks existing in the widespread use of existing Bt toxins, and may replace Bt in the future. It is of great scientific and practical significance to prevent and control the use of pesticides in biological control of agricultural pests.
  • Figure 1 is a schematic diagram of the results of the C7 ELISA assay.
  • Figure 2 is a schematic diagram showing the results of biological assay of C7.
  • Fig. 3 is a schematic diagram showing the death of the third instar larvae of Cnaphalocrocis medinalis after feeding C7, CK+ and CK-soaked rice leaves, respectively.
  • Fig. 4 is a schematic diagram showing the death of third instar larvae of Plutella xylostella after being fed with C7, CK+, CK-soaked cabbage tablets, respectively.
  • a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% glucose were added to the 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin was added to the 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin, and a mass ratio of 1% glucose were added to the 2 ⁇ TY medium.
  • a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% glucose were added to the TYE solid medium.
  • Tween-20 in a volume ratio of 0.05% was added to the PBS solution.
  • Anti-Cry1B polyclonal antibody, BBMV, unrelated anti-idiotype single chain antibody, non-beta type anti-Id The third instar larvae of ScFv, cabbage leaves and Plutella xylostella were provided by the Key Laboratory of Agricultural Product Quality and Safety Control Technology and Standards of the Ministry of Agriculture of Jiangsu Academy of Agricultural Sciences;
  • the humanized phage antibody library, TG1 bacteria and helper phage KM13 were purchased from Source BioScience, UK;
  • HRP-goat anti-M13-IgG was purchased from Wuhan Boster Company;
  • Cry1B toxin and Cry1Ab toxin were purchased from Shanghai Youlong Biotechnology Co., Ltd.;
  • the third instar larvae of rice leaves and leaf roller are provided by Yangzhou Luyuan Biochemical Co., Ltd.
  • the eluate is the phage antibody in the first round of panning; 2, 3,
  • the concentration of the anti-Cry1B polyclonal antibody in the four rounds of panning was 50 ⁇ g/ml, 25 ⁇ g/ml, and 10 ⁇ g/ml, respectively.
  • the phage antibody used was the phage antibody obtained by the previous round of panning, the panning method and the first round.
  • the nucleotide sequence of the selected anti-Cry1B toxin idiotype single-chain antibody was determined by Sanger sequencing method as SEQ ID NO. 1, as follows:
  • the supernatant containing the anti-Cry1B toxin unique type single-chain antibody screened in Example 1 was transferred to 10 ml of 2 ⁇ TY-AG liquid medium at a volume ratio of 1:100, incubated at 37 ° C for 2 h, and the titer of 100 ⁇ l was added.
  • the experiment was divided into 6 experimental groups and corresponding control groups, and the solutions were prepared according to Table 1.
  • C7 is the supernatant of the C7-containing primary culture obtained in Example 2;
  • the experiment was divided into experimental group 1, experimental group 2, experimental group 3, positive control group, negative control group 1, negative control group 2, and negative control group 3; the experimental steps were as follows:
  • C7 is the supernatant of the C7-containing primary culture obtained in Example 2;
  • the experiment was divided into experimental group and control group:
  • the experimental group used the supernatant of C7 primary culture obtained in Example 2 (C7);
  • the positive control group used Cry1Ab toxin (CK+) at a concentration of 0.2 g/L;
  • the negative control group used anti-Id ScFvs (CK-) other than " ⁇ " type;
  • Figure 3 shows the rice vertical rolls after feeding the rice leaves soaked with C7, Cry1Ab toxin (CK+) and non- ⁇ type Anti-Id ScFvs (CK-).
  • CK+ Cry1Ab toxin
  • CK- non- ⁇ type Anti-Id ScFvs

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/CN2015/070421 2014-01-26 2015-01-09 一种人源抗虫基因及其编码的抗Cry1B毒素独特型单链抗体与应用 Ceased WO2015109952A1 (zh)

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Application Number Priority Date Filing Date Title
US15/104,127 US9770038B2 (en) 2014-01-26 2015-01-09 Human-derived insect-resistant gene and anti-CRY1B toxin idiotype single-chain antibody encoded thereby and application thereof
JP2016535243A JP6196382B2 (ja) 2014-01-26 2015-01-09 ヒト耐虫遺伝子およびそれによりコードされた抗Ｃｒｙ１Ｂ毒素イディオタイプ一本鎖抗体ならびに用途
BR112016016014-2A BR112016016014B1 (pt) 2014-01-26 2015-01-09 Gene resistente a inseto derivado de ser humano e anticorpo de cadeia única de idiótipo de anti-toxina cry1b codificado pelo mesmo e sua aplicação
EP15739989.0A EP3098316B1 (en) 2014-01-26 2015-01-09 Human-derived insect-resistant gene and anti-cry1b toxin idiotype single-chain antibody encoded thereby and application thereof

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CN201410037175.X 2014-01-26

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CN103773775B (zh) 2014-01-26 2015-11-18 江苏省农业科学院 一种人源抗虫基因及其编码的抗Cry1B毒素独特型单链抗体与应用
CN104498501B (zh) 2014-12-05 2017-07-18 江苏省农业科学院 一种人源杀虫基因及其编码杀虫肽与应用
CN104926940B (zh) * 2015-06-15 2018-02-09 江苏省农业科学院 一种人源杀虫蛋白及其制备方法与应用
CN105131123B (zh) * 2015-09-16 2018-11-27 江苏省农业科学院 一种具有杀虫活性的牛源化改型抗体及其制备方法与应用
CN105085683B (zh) * 2015-09-16 2018-11-27 江苏省农业科学院 一种具有杀虫活性的猪源化改型抗体及其制备方法与应用
US10981996B1 (en) 2020-06-01 2021-04-20 Abexxa Biologics, Inc. Antibodies targeting a complex comprising non-classical HLA-I and neoantigen and their methods of use
US10981997B1 (en) * 2020-06-01 2021-04-20 Abexxa Biologics, Inc. Antibodies targeting a complex comprising non-classical HLA-I and neoantigen and their methods of use
US11976120B2 (en) 2020-06-01 2024-05-07 Boehringer Ingelheim International Gmbh Antibodies targeting a complex comprising non-classical HLA-I and neoantigen and their methods of use
CN111909264B (zh) * 2020-08-05 2022-02-25 江苏省农业科学院 一种人源化抗金黄色葡萄球菌生物活性肽及其编码基因与应用

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US9770038B2 (en) 2017-09-26
BR112016016014B1 (pt) 2022-06-07
CN103773775B (zh) 2015-11-18
EP3098316A9 (en) 2017-06-28
EP3098316A4 (en) 2017-06-21
EP3098316A1 (en) 2016-11-30
CN103773775A (zh) 2014-05-07
EP3098316B1 (en) 2019-09-18
US20170035055A1 (en) 2017-02-09
JP6196382B2 (ja) 2017-09-13
JP2017505110A (ja) 2017-02-16
BR112016016014A2 (pt) 2017-12-12

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